The present invention relates to a method for detecting substances inhibiting the type III secretion mechanism, which is highly conserved in bacteria belonging to, for example, genus Salmonella, genus Yersinia, genus Pseudomonas, Shigella, Enteropathogenic E. coli, Enterohaemorrhagic E. coli or genus Bordetella, and inhibiting functions of secretory proteins, within short time and large amounts thereof.
Type III secretion mechanism, which functions to release bacterial pathogenic factors extracellularly, is highly conserved, for example, in genus Salmonella, genus Yersinia, genus Pseudomonas, Shigella, Enteropathogenic E. coli (hereinafter sometimes abbreviated as EPEC), Enterohaemorrhagic E. coli and genus Bordetella (Microbiology and Molecular Biology Reviews, June, 1998, p. 381).
It is known that bacteria, which conserves type III secretion mechanism hereinabove, release pathogenic factors extracellularly and a part of the released pathogenic factors was translocated into host cells (Microbiology and Molecular Biology Reviews, June, 1998, p. 389). The translocated pathogenic factors in the host cells are largely involved in pathogenicity of bacteria (Microbiology and Molecular Biology Reviews, June, 1998, p. 382-).
It ha demonstrated by in vivo infection experiments in rabbits (J. Exp. Med. 188(10), 1907-1916, Nov. 16, 1998) and infection tests with human volunteers (Infection and Immunity, June 2000, 3688-3695) that EPEC strain, which defects proteins secreted from the type III secretion mechanism (hereinafter sometimes designates as type III secreted protein), greatly decreased virulence. According to these facts, substances which inhibit the type III secretion mechanism and functions of the secretory proteins are expected their effects as new drugs for treatment and prevention of infectious diseases.
However, no methods for detecting substances which inhibit bacterial type III secretion mechanism have been established, furthermore at present, methods for detection based on antimicrobial activity have been mainstream. Methods for detecting substances having antimicrobial activities, i.e. antibiotic activities, have performed mainly by bioassay. The bioassay includes diffusion method (Microbiological Medicinal Chemistry, Rev. 3rd Ed., Nankodo Publ., Ed. Ohmura, Statoshi). When filter papers containing antibiotics are placed on agar plates containing test microorganisms, the antibiotics diffuse into the agar and inhibitory zone against growth of test microorganisms is observed as transparent zone. The detection method includes calculation of antibiotic potency from relationship between diameters of inhibitory zones and concentration of antibiotics.
Since the diffusion method is simple in operation and large numbers of samples can be treated within short time, it has widely used for detection of antibiotics. However, substances obtained by such method have bactericidal action against not only target microbes but also normal enterobacterial flora, as a result replacement of bacteria and multidrug resistant strains have appeared and at present various problems occur. Consequently, there are problems to apply such the test method for detecting substances which inhibit the bacterial type III secretion. From these facts, methods for detecting substances specifically inhibiting the type III secretion mechanism and the type III secreted protein secreted therefrom have not been established. Consequently, detection of substances, which inhibit the type III secretion mechanism and functions of the type III secretory protein secreted therefrom, have to rely upon experimental animal infective tests. As a result, complex operations have to be required and treatments of large numbers of test samples within short time were difficult.
We have studied that ideas for establishment of simple methods for detecting large amount of substances, which specifically inhibit the type III secretion mechanism and the functions of the type III secreted proteins secreted therefrom within short time, would be very important for development and evaluation of pharmaceutical product targeting the type III secretion mechanism. As a result, we have found novel detection methods without relying upon the experimental tests of animal infection.
An object of the present invention is to provide a method for detecting substances inhibiting the bacterial type III secretion mechanism and the functions of type III secreted proteins secreted therefrom wherein large numbers of the substances specifically inhibiting the bacterial type III secretion mechanism and the functions of the type III secreted proteins secreted therefrom can be detected within short time.
Another object of the present invention is to provide the method for detecting substances inhibiting function of the bacterial type III secretion mechanism and the type III secreted protein secreted therefrom wherein the substances inhibiting the bacterial type III secretion mechanism and the function of the type III secreted proteins secreted therefrom are expressed numerically as an index of the hemolytic activity of erythrocytes and as a result, large numbers of samples can be treated within short time.
When bacteria containing the type III secretion mechanism contacted with erythrocyte, hemolytic activity is induced. It has demonstrated that this hemolytic activity was dependent on an action of the type III secreted proteins on the erythrocytes (Infect. Immun. 67, 5538-5540, 1999). The present invention has completed by observing hemolytic activity induced bacteria containing the type III secretory mechanism. Consequently, the present invention is characterized by the inhibition of hemolytic activity by an addition of substances which inhibit the type III secretion mechanism.
The present invention relates to a method for detecting substances, which inhibit the type III secretion mechanism and the function of the type III secreted proteins secreted therefrom, by employing the hemolytic activity of erythrocytes as an index. The present invention relates to the method for detecting substances which inhibit bacterial type III secretion mechanism comprising mixing the bacteria containing the type III secretion mechanism and erythrocytes suspension, adding the substance which inhibits the type III secretion mechanism and detecting the detectable changes of the generated hemolytic activity. The present invention further relates to the method for detecting substances, which inhibit functions of the secreted proteins of the type III secretion mechanism, comprising mixing the bacteria containing the type III secretion mechanism and erythrocytes suspension, adding the substance, which inhibits the functions of the proteins secreted by an action of the type III secretion mechanism, and detecting the detectable changes of the generated hemolytic activity. Further, the present invention relates to the method for detection wherein the hemolytic activity is detected by colorimetry.
Test microorganisms for inducing hemolytic activities used in the present invention include any bacteria containing the type III secretion mechanism. Examples of bacteria used are, for example, genus Salmonella, genus Yersinia, genus Pseudomonas, Shigella, Enteropathogenic E. coli, Enterohaemorrhagic E. coli and genus Bordetella. In addition to these bacteria, recombinant bacteria, in which DNA regions including gene group, designated as LEE, DNA sequence of 35.4 kbp, coding the type III secretion mechanism and secreted proteins therefrom are artificially integrated into the other bacteria (Molecular Micro. 27, 399-407, 1997) and the similar recombinant bacteria are also included in the present invention.
Growth of test microorganisms is preferably performed in a relatively low nutritional medium such as M9 medium rather than in nutrient rich medium. Culture condition of the test microorganisms is preferably at 37xc2x0 C., and the standing culture is more preferable than the culture with shaking. A principle of hemolytic activity assay is to utilize the fact that when erythrocyte membranes are destroyed, hemoglobins are released into the external erythrocytes to exhibit red color in the reaction medium, and the resulting red color is determined macroscopically. Erythrocytes used in the hemolytic reaction are not limited within human origins, and erythrocytes of any species, for example rabbit, horse, sheep, etc., can be used.